Urinary tract infections (UTIs) are among the most common bacterial infections in the world and result in more than $1.5 billion USD in associated medical costs. The most common cause of UTIs is uropathogenic Escherichia coli (UPEC). UPEC use the chaperone/usher (CU) pathway to assemble and secrete pili onto their cell surfaces. These pili are virulence-associated structures that project away from the cell surface in a hairlike fashion and mediate adhesion to the urinary tract. The chaperone and usher proteins of the CU pathway work in unison to construct these pili. The chaperone, a periplasmic protein, facilitates subunit folding, prevents premature subunit-subunit interactions, and targets subunits to the outer membrane usher protein. The usher is a multifunctional pilus assembly and secretion platform, catalyzing the exchange of chaperone-subunit for subunit-subunit interactions to assemble the pilus fiber and providing the channel for secretion of the fiber to th cell surface. The usher is a large protein consisting of a periplasmic N-terminal domain (N), a transmembrane beta-barrel domain that is gated by an internal Plug domain, and two periplasmic C-terminal domains (C1 and C2). These domains act together to facilitate the ordered assembly and secretion of the pilus. The overall goal of this proposal is to structurally and mechanistically characterize the usher's role in pilus biogenesis. This proposal will test the hypothesis that the usher is both a gated secretion channel and catalytic nanomachine, and that its catalytic activity is due to a carefully coordinated sequence of usher-chaperone-subunit interactions and the accurate placement of chaperone-subunit complexes relative to one another. I propose to use fluorescence techniques to understand how the usher transfers pilus subunits from its N domain to its C domains, a necessary step to clear the N domain for incorporation of the next pilus subunit. I will use similar techniques to understand the Plug's interaction with the C domains of the inactivated usher and how and when the Plug domain is removed from the usher channel upon usher activation. Finally, I propose to probe the structural basis for usher-catalyzed pilus assembly and secretion. I will use molecular techniques to generate pilus assembly intermediates suitable for analysis by cryo-electron microscopy and X-ray crystallography. The information gained by the experiments described in this proposal will lead to new advances in the field of protein secretion. The results will aid in the identification f physical and mechanistic targets for novel therapeutics against UPEC, a particularly important endeavor in the current era of rampant antibiotic resistance.